Mesothelioma cells grow diffusely throughout pleural and peritoneal cavities and do not typically form discreet tumor masses. Consequently, it is difficult to establish the extent of disease and to monitor the response to chemotherapy by conventional radiographic techniques. Mesothelin is a PI-linked glycoprotein that is produced by mesothelial and ovarian cells, but it is not widely produced by other tissues or cells. A significant fraction of mesothelin is known to be shed by tumor cells into culture media and has been previously observed to be elevated in serum from patients with mesothelioma, using a semiquantitative ELISA assay. The objective of this project is to produce a quantitative immunoassay for mesothelin and assess its possible clinical utility. A two-site sandwich type ELISA assay has been developed for mesothelin. The assay has a functional sensitivity of approximately 1 ng/mL and can detect mesothelin in normal subjects. All patients with mesothelioma (N=6) had at least a 3-fold increase in serum mesothelin compared to normal subjects. Approximately 80% of ovarian cancer patients (N=20) also had elevated levels of serum mesothelin. Mesothelin was not observed to be increased in random hospitalized patients (N=40). In the upcoming year, we plan to do the following: (1) Develop a more sensitive and precise mesothelin assay, using an automated chemiluminescent immunoassay system. (2) Establish a reference range for normal subjects. (3) Establish the typical range of serum mesothelin in patients with mesothelioma and ovarian cancer. (4) Test the specificity of the assay, as a tumor marker, by measuring mesothelin in a large number of random hospitalized patients and in patients with non-malignant pulmonary and GI disorders. (5) Characterize the molecular forms of serum mesothelin by Western blot/MALDI-TOF Mass spectroscopy. (6) Determine the response of serum mesothelin to chemotherapy in patients with mesothelioma and ovarian cancer.